Strain improvement of <Emphasis Type="Italic">Lactobacillus lactis</Emphasis> for <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid production

Three mutants, isolated by repeated UV mutagenesis of Lactobacillus lactis NCIM 2368, produced increased d-lactic acid concentrations. These mutants were compared with the wild type using 100 g hydrolyzed cane sugar/l in the fermentation medium. One mutant, RM2-24, produced 81 g lactic acid/l which was over three times that of the wild type. The highest d-lactic acid (110 g/l) in batch fermentation was obtained with 150 g cane sugar/l with a 73% lactic acid yield. The mutant utilizes cellobiose efficiently, converting it into d-lactic acid suggesting the presence of cellobiase. Thus, this strain could be used to obtain d-lactic acid from cellulosic materials that are pre-hydrolyzed with cellulase.     

Continuous <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid production by a novelthermotolerant <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> QU 41

We isolated and characterized a d-lactic acid-producing lactic acid bacterium (d-LAB), identified as Lactobacillus delbrueckii subsp. lactis QU 41. When compared to Lactobacillus coryniformis subsp. torquens JCM 1166 ^T and L. delbrueckii subsp. lactis JCM 1248 ^T, which are also known as d-LAB, the QU 41 strain exhibited a high thermotolerance and produced d-lactic acid at temperatures of 50 °C and higher. In order to optimize the culture conditions of the QU 41 strain, we examined the effects of pH control, temperature, neutralizing reagent, and initial glucose concentration on d-lactic acid production in batch cultures. It was found that the optimal production of 20.1 g/l d-lactic acid was acquired with high optical purity (>99.9% of d-lactic acid) in a pH 6.0-controlled batch culture, by adding ammonium hydroxide as a neutralizing reagent, at 43 °C in MRS medium containing 20 g/l glucose. As a result of product inhibition and low cell density, continuous cultures were investigated using a microfiltration membrane module to recycle flow-through cells in order to improve d-lactic acid productivity. At a dilution rate of 0.87 h⁻¹, the high cell density continuous culture exhibited the highest d-lactic acid productivity of 18.0 g/l/h with a high yield (ca. 1.0 g/g consumed glucose) and a low residual glucose (<0.1 g/l) in comparison with systems published to date.     

Highly efficient production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid from chicory-derived inulin by <Emphasis Type="Italic">Lactobacillus bulgaricus</Emphasis>

Inulin is a readily available feedstock for cost-effective production of biochemicals. To date, several studies have explored the production of bioethanol, high-fructose syrup and fructooligosaccharide, but there are no studies regarding the production of d-lactic acid using inulin as a carbon source. In the present study, chicory-derived inulin was used for d-lactic acid biosynthesis by Lactobacillus bulgaricus CGMCC 1.6970. Compared with separate hydrolysis and fermentation processes, simultaneous saccharification and fermentation (SSF) has demonstrated the best performance of d-lactic acid production. Because it prevents fructose inhibition and promotes the complete hydrolysis of inulin, the highest d-lactic acid concentration (123.6 ± 0.9 g/L) with a yield of 97.9 % was obtained from 120 g/L inulin by SSF. Moreover, SSF by L. bulgaricus CGMCC 1.6970 offered another distinct advantage with respect to the higher optical purity of d-lactic acid (>99.9 %) and reduced number of residual sugars. The excellent performance of d-lactic acid production from inulin by SSF represents a high-yield method for d-lactic acid production from non-food grains.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid by <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> under oxygen deprivation

In mineral salts medium under oxygen deprivation, Corynebacterium glutamicum exhibits high productivity of l-lactic acid accompanied with succinic and acetic acids. In taking advantage of this elevated productivity, C. glutamicum was genetically modified to produce d-lactic acid. The modification involved expression of fermentative d-lactate dehydrogenase (d-LDH)-encoding genes from Escherichia coli and Lactobacillus delbrueckii in l-lactate dehydrogenase (l-LDH)-encoding ldhA-null C. glutamicum mutants to yield strains C. glutamicum ΔldhA/pCRB201 and C. glutamicum ΔldhA/pCRB204, respectively. The productivity of C. glutamicum ΔldhA/pCRB204 was fivefold higher than that of C. glutamicum ΔldhA/pCRB201. By using C. glutamicum ΔldhA/pCRB204 cells packed to a high density in mineral salts medium, up to 1,336 mM (120 g l⁻¹) of d-lactic acid of greater than 99.9% optical purity was produced within 30 h.     

Biotechnological production of enantiomerically pure <Emphasis Type="Italic">d</Emphasis>-lactic acid

The fermentation process of l-lactic acid is well known. Little importance was attached to d-lactic acid, but in the past 10 years, d-lactic acid gained significantly in importance. d-Lactic acid is an interesting precursor for manufacturing heat-resistant polylactic acid (PLA) bioplastics which can be widely used, for example as packaging material, coatings, for textiles or in the automotive industry.This review provides a comprehensive overview of the most recent developments, including a spectrum of studied microorganisms and their capabilities for the production of d-lactic acid. Additionally, the technological achievements in biotechnological d-lactic acid production including fermentation techniques like fed batch, simultaneous saccharification, and fermentation and continuous techniques are presented. Attention is also turned to suitable alternative substrates and their applicability in fermentation processes. Furthermore, advantages and disadvantages of product recovery and purification are discussed. Economic aspects of PLA are pointed out, and the present industrial producers of lactic acid are briefly introduced.     

Biosynthesis of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid from lignocellulosic biomass

d-lactic acid is a versatile and important industrial chemical that can be applied in the synthesis of thermal-resistant poly-lactic acid. Biosynthesis of d-lactic acid can be achieved by a variety of microorganisms, including lactic acid bacteria, yeast, and fungi; however, the final product yield, optical purity, and the utilization of both glucose and xylose are restricted. Consequently, engineered microbial systems are essential to attain high titer, productivity, and complete utilization of sugars. Herein, we critically evaluate the promising wild-type microorganisms, as well as genetically modified microorganisms to produce enantiomerically pure d-lactic acid, particularly from renewable lignocellulosic biomass. In addition, innovative bioreactor operation, metabolic flux analysis, and recent genetic engineering methods for targeted microbial d-lactic acid synthesis will be discussed.     

<Emphasis Type="SmallCaps">d</Emphasis>-Psicose production from <Emphasis Type="SmallCaps">d</Emphasis>-fructose using an isolated strain, <Emphasis Type="Italic">Sinorhizobium</Emphasis> sp.

Sinorhizobium sp., which can convert d-fructose into d-psicose, was isolated from soil. The optimal pH, temperature, and cell concentration for d-psicose production with the isolated strain were 8.5, 40°C, and 60 mg/ml, respectively. The toluene-treated cells showed 2.5- and 4.8-fold increases in the d-psicose concentration and productivity compared with untreated washed cells. Under the optimal conditions, the toluene-treated cells produced 37 g d-psicose/l from 70% (w/v) (3.9 M) d-fructose after 15 h.     

Purification and characterization of <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-tagatose

l-arabinose isomerase (EC5.3.1.4. AI) mediates the isomerization of d-galactose into d-tagatose as well as the conversion of l-arabinose into l-ribulose. The AI from Lactobacillus plantarum SK-2 was purified to an apparent homogeneity giving a single band on SDS–PAGE with a molecular mass of 59.6 kDa. Optimum activity was observed at 50°C and pH 7.0. The enzyme was stable at 50°C for 2 h and held between pH 4.5 and 8.5 for 1 h. AI activity was stimulated by Mn²⁺, Fe³⁺, Fe²⁺, Ca²⁺ and inhibited by Cu²⁺, Ag⁺, Hg²⁺, Pb²⁺. d-galactose and l-arabinose as substrates were isomerized with high activity. l-arabitol was the strongest competitive inhibitor of AI. The apparent Michaelis–Menten constant (K _m), for galactose, was 119 mM. The first ten N-terminal amino acids of the enzyme were determined as MLSVPDYEFW, which is identical to L. plantarum (Q88S84). Using the purified AI, 390 mg tagatose could be converted from 1,000 mg galactose in 96 h, and this production corresponds to a 39% equilibrium.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid from sugarcane molasses,sugarcane juice and sugar beet juice by <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis>

Lactobacillus delbrueckii was grown on sugarcane molasses, sugarcane juice and sugar beet juice in batch fermentation at pH 6 and at 40°C. After 72 h, the lactic acid from 13% (w/v) sugarcane molasses (119 g total sugar l⁻¹) and sugarcane juice (133 g total sugar l⁻¹) was 107 g l⁻¹ and 120 g l⁻¹, respectively. With 10% (w/v) sugar beet juice (105 g total sugar l⁻¹), 84 g lactic acid l⁻¹ was produced. The optical purities of d-lactic acid from the feedstocks ranged from 97.2 to 98.3%.     

Fermentation of <Emphasis Type="SmallCaps">d</Emphasis>-glucose and <Emphasis Type="SmallCaps">d</Emphasis>-xylose mixtures by <Emphasis Type="Italic">Candida tropicalis</Emphasis> NBRC 0618 for xylitol production

The fermentation of d-glucose and d-xylose mixtures by the yeast Candida tropicalis NBRC 0618 has been studied under the most favourable operation conditions for the culture, determining the most adequate initial proportion in these sugars for xylitol production. In all the experiments a synthetic culture medium was used, with an initial total substrate concentration of 25 g L⁻¹, a constant pH of 5.0 and a temperature of 30 °C. From the experimental results, it was deduced that the highest values of specific rates of production and of overall yield in xylitol were achieved for the mixtures with the highest percentage of d-xylose, specifically in the culture with the initial d-glucose and d-xylose concentrations of 1 and 24 g L⁻¹, respectively, with an overall xylitol yield of 0.28 g g⁻¹. In addition, the specific rates of xylitol production declined over the time course of the culture and the formation of this bioproduct was favoured by the presence of small quantities of d-glucose. The sum of the overall yield values in xylitol and ethanol for all the experiments ranged from 0.26 to 0.56 g bioproduct/g total substrate.     

Characterization of <Emphasis Type="SmallCaps">d</Emphasis>-tagatose-3-epimerase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> that converts <Emphasis Type="SmallCaps">d</Emphasis>-fructose into <Emphasis Type="SmallCaps">d-</Emphasis>psicose

A non-characterized gene, previously proposed as the d-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with d-fructose and decreased for other substrates in the order: d-tagatose, d-psicose, d-ribulose, d-xylulose and d-sorbose. Its activity was maximal at pH 9 and 40°C while being enhanced by Mn²⁺. At pH 9 and 40°C, 118 g d-psicose l⁻¹ was produced from 700 g d-fructose l⁻¹ after 3 h. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of ribose-5-phosphate isomerase of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-allose from <Emphasis Type="SmallCaps">d</Emphasis>-psicose

The rpiB gene, encoding ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum, was cloned and expressed in Escherichia coli. RpiB converted d-psicose into d-allose but it did not convert d-xylose, l-rhamnose, d-altrose or d-galactose. The production of d-allose by RpiB was maximal at pH 7.5 and 65°C for 30 min. The half-lives of the enzyme at 50°C and 65°C were 96 h and 4.7 h, respectively. Under stable conditions of pH 7.5 and 50°C, 165 g d-allose l⁻¹ was produced without by-products from 500 g d-psicose l⁻¹ after 6 h.     

Phosphoenolpyruvate-supply module in <Emphasis Type="Italic">Escherichia coli</Emphasis> improves <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">d</Emphasis>-neuraminic acid biocatalysis

Objectives

N-Acetyl-d-neuraminic acid (Neu5Ac) is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and excess pyruvate. We have previously constructed a recombinant Escherichia coli strain for Neu5Ac production using GlcNAc and intracellular phosphoenolpyruvate (PEP) as substrates (Zhu et al. Biotechnol Lett 38:1–9, 2016).

Results

PEP synthesis-related genes, pck and ppsA, were overexpressed within different modes to construct PEP-supply modules, and their effects on Neu5Ac production were investigated. All the PEP-supply modules enhanced Neu5Ac production. For the best module, pCDF-pck-ppsA increased Neu5Ac production to 8.6 ± 0.15 g l^?1, compared with 3.6 ± 0.15 g l^?1 of the original strain. Neu5Ac production was further increased to 15 ± 0.33 g l^?1 in a 1 l fermenter.

Conclusions

The PEP-supply module can improve the intracellular PEP supply and enhance Neu5Ac production, which benefited industrial Neu5Ac production.

     

<Emphasis Type="SmallCaps">d</Emphasis>-Hexosaminate production by oxidative fermentation

Microbial production of d-hexosaminate was examined by means of oxidative fermentation with acetic acid bacteria. In most strains of acetic acid bacteria, membrane-bound d-glucosamine dehydrogenase (synonymous with an alternative d-glucose dehydrogenase distinct from quinoprotein d-glucose dehydrogenase) oxidized d-hexosamines to the corresponding d-hexosaminates in a stoichiometric manner. Conversion of d-hexosamines to the corresponding d-hexosaminates was observed with growing cells of acetic acid bacteria, and d-hexosaminate was stably accumulated in the culture medium even though d-hexosamine was exhausted. Since the enzyme responsible is located on the outer surface of the cytoplasmic membrane, and the enzyme activity is linked to the respiratory chain of the organisms, resting cells, dried cells, and immobilized cells of acetic acid bacteria were effective catalysts for d-hexosaminate production. d-Mannosaminate and d-galactosaminate were also prepared for the first time by means of oxidative fermentation, and three different d-hexosaminates were isolated from unreacted substrate by a chromatographic separation. In this paper, d-hexosaminate production by oxidative fermentation carried out mainly with Gluconobacter frateurii IFO 3264 is exemplified as a typical example.     

Biotransformation of glycerol to <Emphasis Type="SmallCaps">d</Emphasis>-glyceric acid by <Emphasis Type="Italic">Acetobacter tropicalis</Emphasis>

Bacterial strains capable of converting glycerol to glyceric acid (GA) were screened among the genera Acetobacter and Gluconacetobacter. Most of the tested Acetobacter and Gluconacetobacter strains could produce 1.8 to 9.3 g/l GA from 10% (v/v) glycerol when intact cells were used as the enzyme source. Acetobacter tropicalis NBRC16470 was the best GA producer and was therefore further investigated. Based on the results of high-performance liquid chromatography analysis and specific rotation, the enantiomeric composition of the produced GA was d-glyceric acid (d-GA). The productivity of d-GA was enhanced with the addition of both 15% (v/v) glycerol and 20 g/l yeast extract. Under these optimized conditions, A. tropicalis NBRC16470 produced 22.7 g/l d-GA from 200 g/l glycerol during 4 days of incubation in a jar fermentor.     

Removal of <Emphasis Type="SmallCaps">l</Emphasis>-alanine from the production of <Emphasis Type="SmallCaps">l</Emphasis>-2-aminobutyric acid by introduction of alanine racemase and <Emphasis Type="SmallCaps">d</Emphasis>-amino acid oxidase

l-2-Aminobutyric acid can be synthesized in a transamination reaction from l-threonine and l-aspartic acid as substrates by the action of threonine deaminase and aromatic aminotransferase, but the by-product l-alanine was produced simultaneously. A small amount of l-alanine increased the complexity of the l-2-aminobutyric acid recovery process because of their extreme similarity in physical and chemical properties. Acetolactate synthase has been introduced to remove the pyruvate intermediate for reducing the l-alanine concentration partially. To eliminate the remnant l-alanine, alanine racemase of Bacillus subtilis in combination with d-amino acid oxidase of Rhodotorula gracilis or Trigonopsis variabilis respectively was introduced into the reaction system for the l-2-aminobutyric acid synthesis. l-Alanine could be completely removed by the action of alanine racemase of B. subtilis and d-amino acid oxidase of R. gracilis; thereby, high-purity l-2-aminobutyric acid was achieved. The results revealed that alanine racemase could discriminate effectively between l-alanine and l-2-aminobutyric acid, and selectively catalyzed l-alanine to d-alanine reversibly. d-Amino acid oxidase then catalyzed d-alanine to pyruvate stereoselectively. Furthermore, this method was also successfully used to remove the by-product l-alanine in the production of other neutral amino acids such as l-tertiary leucine and l-valine, suggesting that multienzymatic whole-cell catalysis can be employed to provide high purity products.     

Improved xylanase production by<Emphasis Type="Italic"> Trichoderma reesei</Emphasis> grown on <Emphasis Type="SmallCaps">l</Emphasis>-arabinose and lactose or <Emphasis Type="SmallCaps">d</Emphasis>-glucose mixtures

Trichoderma reesei Rut C-30 was grown on eight different natural or rare aldopentoses as the main carbon source and on mixtures of an aldopentose with d-glucose or lactose. The fungal cells consumed all aldopentoses tested, except l-xylose and l-ribose. The highest total xylanase and cellulase activities were achieved when cells were grown on l-arabinose as the main carbon source. The total xylanase activity produced by cells grown on l-arabinose was even higher than that produced by cells grown on an equal amount of lactose. In co-metabolism of d-glucose (15 g l^–1) and l-arabinose (5 g l^–1), the total volumetric and specific xylanase productivities were improved (derepressed) approximately 23- and 18-fold, respectively, compared to a cultivation on only d-glucose (20 g l^–1). In a similar experiment, in which cells were grown on a mixture of lactose and l-arabinose, the xylanase productivity was approximately doubled, compared to a cultivation on only lactose. The cellulase productivities, however, were not improved by the addition of l-arabinose. Compared with a typical industrial fungal enzyme production process with lactose as the main carbon source, better volumetric and specific xylanase productivities were achieved both on a lactose/arabinose mixture and on a glucose/arabinose mixture.     

Hairy root culture optimization and resveratrol production from <Emphasis Type="Italic">Vitis vinifera</Emphasis> subsp. <Emphasis Type="Italic">sylvesteris</Emphasis>

Resveratrol is a polyphenolic compound produced in very low levels in grapes. To achieve high yield of resveratrol in wild grape, three Agrobacterium rhizogenes strains, Ar318, ArA4 and LBA9402, were used to induce hairy roots following infection of internodes, nodes or petioles of in vitro grown Vitis vinifera subsp. sylvesteris accessions W2 and W16, and cultivar Rasha. The effects of inoculation time, age of explants, bacterial concentration and co-cultivation times were examined on the efficiency of the production of hairy roots. Strains Ar318, ArA4 and LBA9402 all induced hairy roots in the tested genotypes, but the efficiency of ArA4 strain was higher than the other strains. The highest hairy root production was with using internodes as explants. The transformation of hairy roots lines was confirmed by PCR detection of rolB gene. Half Murashige and Skoog (MS) medium was better for biomass production compared with MS medium. HPLC analysis of resveratrol production in the hairy root cultures showed that all the genotypes produced higher amounts of resveratrol than control roots. The highest amount of resveratrol was produced from W16 internode cultures, which was 31-fold higher than that of control root. Furthermore, TLC analysis showed that treatments of hairy roots with sodium acetate and jasmonate elevated resveratrol levels both in hairy root tissue and excreted into the half MS medium. These results demonstrate that endogenous and exogenous factors can affect resveratrol production in hairy root culture of grape, and this strategy could be used to increase low resveratrol production in grapes.     

An optimized fed-batch culture strategy integrated with a one-step fermentation improves <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid production by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis>

In previous work, we proposed a novel modified one-step fermentation fed-batch strategy to efficiently generate l-lactic acid (l-LA) using Rhizopus oryzae. In this study, to further enhance efficiency of l-LA production through one-step fermentation in fed-batch cultures, we systematically investigated the initial peptone- and glucose-feeding approaches, including different initial peptone and glucose concentrations and maintained residual glucose levels. Based on the results of this study, culturing R. oryzae with initial peptone and glucose concentrations of 3.0 and 50.0 g/l, respectively, using a fed-batch strategy is an effective approach of producing l-LA through one-step fermentation. Changing the residual glucose had no obvious effect on the generation of l-LA. We determined the maximum LA production and productivity to be 162 g/l and 6.23 g/(l·h), respectively, during the acid production stage. Compared to our previous work, there was almost no change in l-LA production or yield; however, the productivity of l-LA increased by 14.3%.     

Biotechnological production of <Emphasis Type="SmallCaps">d</Emphasis>-glyceric acid and its application

Glycerol is currently produced in large amounts as a by-product during fat splitting and biodiesel fuel production. Over the past decade, both chemical and biotechnological processes to convert glycerol to value-added chemicals have been increasingly explored. This mini-review provides recent information about the biotechnological production of a glycerol derivative, d-glyceric acid (d-GA), and its possible applications. Little is known about GA as a bioproduct, but it is naturally found in different kinds of plants as a phytochemical constituent and is reported to have some biological activity. A racemic mixture of dl-GA can be obtained from glycerol via chemical oxidation; however, d-GA is mainly biotechnologically produced with the aid of bacteria. Under aerobic conditions, some acetic acid bacteria transform glycerol into d-GA, and optimization of initial glycerol concentration and aeration rate provided a yield of more than 80 g/l d-GA, using a strain of Gluconobacter frateurii.